1. Field of the Invention
This invention relates to a polypeptide containing at least a specific antigenic region of a human centromere antigen recognized by a human anti-centromere antibody, a gene encoding the polypeptide, a plasmid or a phage containing the gene, a transformant obtained by transforming a host with the plasmid or the phage, a method for producing the human centromere antigen polypeptide utilizing the transformant and a method for detecting an anti-centromere antibody using the human centromere antigen polypeptide.
2. Description of the Prior Art
Autoimmune diseases are among the nationally designated incurable diseases in Japan, and it is required to elucidate causes of the diseases and develop therapeutic methods. Various antibodies against various own cellular components (autoantibodies) are present in the serum of an autoimmune disease patient. It is thought there is an association of these autoantibodies with the diseases.
An anti-centromere antibody is one of the anti-nuclear antibodies present in the central region of a mitotic cell chromosome and reacts with a protein of "a centromere domain" which plays an important role in the separation of a chromosome in a mitotic phase. An anti-centromere antibody has been pointed out as an autoantibody related to some autoimmune diseases, particularly scleroderma. Scleroderma is a general name for the disease inclusive of various disease types, and a method of clinical assay for an easier and more precise classification of the disease type is required to be established in order to diagnose and treat the disease.
An indirect fluorescent antibody technique is currently employed for detecting an anti-centromere antibody in a patient's serum using a liver section or cultured cells as nuclear materials. In order to strictly distinguish the positive antibody from other antibodies present in the serum in the indirect fluorescent antibody method, it should be further confirmed that the anti-centromere antibody recognizes the centromere region on a chromosome further utilizing a chromosome smear of cells in a mitotic phase. The method has disadvantages in that it requires a chromosome smear preparation and a specific facility and labors for microscopic observation of the smear, so that it is not suitable for a clinical assay handling a large number of people. For an alternative method for detecting the anti-centromere antibody activity, a method using a chromosomal centromere region as an antigen prepared from human cells in large quantity is assumed, but it is still too costly and labor intensive for practical use.
Presence of human centromere antigens, each of them including centromere protein A, B or C of different molecular weight has been identified. Among them, the major antigen is human centromere protein B (CENP-B), a portion of the gene coding for the protein has been cloned [Earnshaw, W. C., et al., J. Cell Biol., 104: 817 (1987)]. However, the epitopes contained in CENP-B have not been analyzed in detail yet. For the precise detection and classification of an anti-centromere antibody, detailed analysis of epitopes of the major antigen CENP-B is desired.
In order to solve the conventional problems described above and to develop a method for measuring anti-centromere antibody specifically as well as readily, elucidation of the epitopes of the major human centormere antigen, human centromere protein B and the production of the polypeptides corresponding to each epitope in a large quantity are desired.